All of the cells in the peripheral blood are generated from a small population of hematopoietic stem cells (HSC) through a process of proliferation and differentiation known as hematopoiesis. The hematopoietic differentiation program includes well-defined stages at which the progeny of HSC become restricted to specific fates. The goal of this project is to define specific molecular signatures associated with specific stages of hematopoiesis. Project 1. DNA methylation is an essential epigenetic mark that is required for normal development and hematopoietic differentiation. We characterized genome-wide DNA methylation in primary mouse HSC, Common Myeloid progenitors (CMP), and erythroblasts (ERY). We found that DNA methylation was most abundant in HSC, with a 40% reduction in the number of peaks present in CMP, and a 67% reduction in the number of peaks present in ERY. Over 97% of the peaks present in CMP and ERY were also present in HSC, while only 3% of peaks arise de novo during differentiation. These results demonstrate that the methylation profile of erythroblasts is present in HSC and that a specific genome-wide decrease in DNA methylation occurs during erythropoiesis. (Hogart A, Lichtenberg J, Ajay SS, Anderson S, NIH Intramural Sequencing Center, Margulies EH, Bodine DM. Genome-wide DNA methylation profiles in hematopoietic stem and progenitor cells reveal overrepresentation of ETS transcription factor binding sites. Genome Res. 22 (8) 1407-18, 2012.) The magnitude of the Methyl-Seq data required the development of a novel software package we call SigSeeker. We used SigSeeker in a horizontal analysis of methylation in monocytic cells before and after treatment with 5-aza-cytidine, a DNA methylation inhibitor. (Lichtenberg J, Hogart A, Battle S. Bodine DM. Discovery of motif-based regulatory signatures in whole genome methylation experiments. Bioinformatics Open Source Conference (BOSC), 2012, Long Beach USA.) We found that SigSeeker was equally capable of analyzing ChIP-Seq data, demonstrating the applicability of the tool to many large-scale systems biology problems. (Dworkin AM, Lee M, Lichtenberg J, Patel SJ, Gildea D, Sakthianadeswaren A, NISC Comparative Sequencing Program, Foote S, Wolfsberg TG, Bodine DM, Crawford NPS. The metastasis suppressor RRP1B interacts with TRIM28 and HP1&#945; at the c-MYC locus to induce heterochromatinization and silencing of c-MYC expression. PNAS submitted.) Our current plans are to extend these studies to generate methylation profiles for at least two additional cell types, the Megakaryocyte/Erythroid progenitor (MEP; the progenitor that gives rise to both the erythroid and megakaryocytic platelet lineages) and fully differentiated megakaryocytes. Through these studies we anticipate that we will identify methylation changes that are specific to the erythroid and megakaryocytic (platelet) lineages. Project 2. Diamond Blackfan anemia is an inherited bone marrow failure syndrome characterized by a severe deficiency of red blood cells, despite the fact that all other hematopoietic lineages differentiate normally and are present in normal numbers. Mutations in 9 different ribosomal protein genes have been identified in multiple unrelated families, most resulting in haploinsufficiency. However, 40% of DBA-associated mutations are missense mutations that cause a single amino acid change. These can be divided into two classes. The first type of amino acid substitution (Class I) interferes with the proper folding of the ribosomal protein, resulting in endosomal degradation and functional haploinsufficiency. The second type (Class II) produces proteins that appear to be folded normally as they are stable in the cytoplasm. We hypothesized that the Class II mutations act by a dominant negative mechanism. To test this hypothesis, we generated a transgenic mouse model that expresses an RPS19 mutation at codon 62 (RPS19R62W). Conditional expression of RPS19R62W in hematopoietic cells resulted in anemia with reduced numbers of erythroid progenitors. We concluded that RPS19R62W is a dominant negative DBA mutation. (Devlin EE, Dacosta L, Mohandas N, Elliott G, Bodine DM. A transgenic mouse model demonstrates a dominant negative effect of a point mutation in the RPS19 gene associated with Diamond-Blackfan anemia. Blood. 116 (15): 2826-35, 2010.) Attempts by others to generate mouse models of DBA by knocking out the mouse Rps19 gene have been unsuccessful. Preliminary data suggests that the output of the normal mouse Rps19 allele increases to compensate for the loss of the second allele. RPS19s only function appears to be in the assembly of the small ribosomal subunit. We hypothesized that mild deficiencies in the levels of ribosomal proteins that serve additional functions might be better candidates for a mouse model of DBA. Mutations in the RPL11 gene account for 15% of known DBA mutations. In addition to its role in the assembly of the large ribosomal subunit, RPL11 also interacts with MDM2, p53 and the rRNA processing pathways, which may account for the significantly higher frequency of craniofacial and physical abnormalities seen in patients with RPL11 mutations. We are currently generating an Rpl11 conditional knockout mouse to test this hypothesis and to provide an in vivo system to study the mechanism of erythroid failure and to test potentially useful drugs. Project 3. The goal of this project is to provide a molecular diagnosis to each DBA patient. We hypothesized that copy number variations that cause the deletion of a ribosomal protein gene could cause DBA in patients with normal ribosomal protein gene sequences. We used genome-wide SNP array profiling to evaluate regions of copy variation in DBA patients. In our initial sampling, we identified deletions at known DBA-related ribosomal protein gene loci in 20% of 50 DBA patients. These data suggest that ribosomal protein gene deletions are more common than previously suspected and are a significant cause of DBA. (Farrar JE, Vlachos A, Atsidaftos E, Carlson-Donohoe H, Markello TC, Arceci RJ, Ellis SR, Lipton JM, Bodine DM. Ribosomal protein gene deletions in Diamond-Blackfan Anemia. Blood. 118 (26): 6943-51, 2011). Using a sensitive new method to detect mosaic copy number variations (Markello TC, Carlson-Donohoe H, Sincan M, Adams D, Bodine DM, Farrar JE, Vlachos A, Lipton JM, Auerbach AD, Ostrander EA, Chandrasekharappa SC, Boerkoel CF, Gahl WA. Sensitive quantification of mosaicism using high density SNP arrays and the cumulative distribution function. Mol Genet Metab. 105 (4): 665-71, 2012.), we also identified novel variable mosaic loss involving chromosomal regions containing known DBA gene regions in 5 patients from 4 different kindreds. To identify novel DBA mutations, we plan to expand our sequence analysis of those patients who have not had mutations in known DBA genes identified by sequencing and do not have copy number variations identified by SNP-CGH. We will enroll 4 unrelated family groups consisting of parents, a DBA proband and either an affected or unaffected sibling for exome capture sequencing. The inclusion of the parents and sibling will add power to the analysis of the exome sequence. After the first round of sequencing we will screen the remaining pool of undiagnosed DBA patients for any newly identified mutation(s) before enrolling a second set of 4 families who do not carry a known mutation.